The yeast Saccharomyces cerevisiae can use alternative nitrogen sources such as arginine, urea, allantoin, ␥-aminobutyrate, or proline when preferred nitrogen sources like glutamine, asparagine, or ammonium ions are unavailable in the environment. Utilization of alternative nitrogen sources requires the relief of nitrogen repression and induction of specific permeases and enzymes. The products of the GLN3 and URE2 genes are required for the appropriate transcription of many genes in alternative nitrogen assimilatory pathways. GLN3 appears to activate their transcription when good nitrogen sources are unavailable, and URE2 appears to repress their transcription when alternative nitrogen sources are not needed. The participation of nitrogen repression and the regulators GLN3 and URE2 in the proline utilization pathway was evaluated in this study.Comparison of PUT gene expression in cells grown in repressing or derepressing nitrogen sources, in the absence of the inducer proline, indicated that both PUT1 and PUT2 are regulated by nitrogen repression, although the effect on PUT2 is comparatively small. Recessive mutations in URE2 elevated expression of the PUT1 and PUT2 genes 5-to 10-fold when cells were grown on a nitrogen-repressing medium. Although PUT3, the proline utilization pathway transcriptional activator, is absolutely required for growth on proline as the sole nitrogen source, a put3 ure2 strain had somewhat elevated PUT gene expression, suggesting an effect of the ure2 mutation in the absence of the PUT3 product. PUT1 and PUT2 gene expression did not require the GLN3 activator protein for expression under either repressing or derepressing conditions. Therefore, regulation of the PUT genes by URE2 does not require a functional GLN3 protein. The effect of the ure2 mutation on the PUT genes is not due to increased internal proline levels. URE2 repression appears to be limited to nitrogen assimilatory systems and does not affect genes involved in carbon, inositol, or phosphate metabolism or in mating-type control and sporulation.The proline utilization pathway in Saccharomyces cerevisiae enables cells to use proline as the sole source of nitrogen when preferred nitrogen sources are not available in the environment. The proline utilization enzymes proline oxidase and ⌬ 1 -pyrroline-5-carboxylate dehydrogenase, encoded by the nuclear genes PUT1 and PUT2, respectively, convert proline to glutamate in mitochondria (9-11). The expression of the PUT genes is regulated by the PUT3 activator protein, which responds to the presence of proline in the medium and increases the transcription of the PUT1 and PUT2 genes (7,8,67). The PUT3 protein constitutively binds to the upstream activation sequences in the promoters of both PUT1 and PUT2 genes in vitro and in vivo but activates transcription only in the presence of proline (2,44,45,58).Early studies on the proline utilization pathway concluded that the structural genes PUT1 and PUT2 were not regulated by nitrogen repression (10). We speculated that the proline transpor...

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In Saccharomyces cerevisiae, the ability to use proline as a nitrogen source requires the Put3p transcriptional regulator, which turns on the expression of the proline utilization genes, PUT1 and PUT2, in the presence of the inducer proline and in the absence of preferred nitrogen sources. Changes in target gene expression occur through an alteration in activity of the DNA‐bound Put3p, a member of the Zn(II)2Cys6 binuclear cluster family of proteins. Here, we report that the ‘on’ conformation can be mimicked in the absence of proline by the insertion of an epitope tag in several different places in the protein, as well as by specific amino acid changes that suppress a put3 mutation leading to non‐inducibility of the pathway. In addition, the presence of proline causes a conformational change in the Put3 protein detected by increased sensitivity to thrombin or V8 protease. These findings suggest that Put3p shifts from an inactive to an activate state via conformational changes.

This report describes a transfection-independent system for packaging alphavirus replicon vectors using modified vaccinia virus Ankara (MVA) vectors to express all of the RNA components necessary for the production of Venezuelan equine encephalitis (VEE) virus replicon particles (VRP). Infection of mammalian cells with these recombinant MVA vectors resulted in robust expression of VEE structural genes, replication of the alphavirus vector and high titers of VRP. In addition, VRP packaging was achieved in a cell type (fetal rhesus lung) that has been approved for the manufacturing of vaccines destined for human use.

The PRO3 gene of Saccharomyces cerevisiae encodes the 286-amino-acid protein AW-pyrroline-5-carboxylate reductase [L-proline:NAD(P+) 5-oxidoreductase; EC 1.5.1.21, which catalyzes the final step in proline biosynthesis. The protein has substantial similarity to the pyrroline carboxylate reductases of diverse bacterial species, soybean, and humans. Using RNA hybridization and measurements of enzyme activity, we have determined that the expression of the PRO3 gene appears to be constitutive. It is not repressed by the pathway end product (proline), induced by the initial substrate (glutamate), or regulated by the general control system. Its expression is not detectably altered when cells are grown in a wide range of nitrogen sources or when glycerol and ethanol replace glucose as the carbon source. The possibility that this enzyme has other functions in addition to proline biosynthesis is discussed.In the yeast Saccharomyces cerevisiae, the pathway of proline biosynthesis was established by characterization of proline-requiring mutants and interspecies complementation of Escherichia coli and Salmonella typhimurium proline auxotrophs by the cloned yeast genes (6,38 In addition to the formation of proline required for protein synthesis, the final step in the proline biosynthetic pathway generates NADP+, which is believed to stimulate the production of ribose-5-phosphate via the pentose phosphate shunt in human erythrocytes (42) and in soybean nodules (22). This stimulation results in an increase in 5-phosphoribosyl 1-pyrophosphate, leading to an increase in de novo purine biosynthesis. Because of the variety of metabolic interconversions of proline and P5C, these compounds link the tricarboxylic acid and urea cycles with amino acid and purine biosynthesis in a diversity of cell types.With this report, we begin a molecular analysis of the S. cerevisiae gene encoding P5C reductase, the enzyme that catalyzes the final step of proline biosynthesis. Because arginine catabolism and proline biosynthesis converge at P5C (10), P5C reductase is both a proline-biosynthetic and an arginine-degradative enzyme. A biochemical analysis of the purified protein (26, 27) and its cytosolic localization (11) * Corresponding author.were reported a decade ago. pro3 mutants require proline for growth, lack P5C reductase activity, and have the peculiar property of being unable to grow on rich, or completely supplemented, minimal media (6,38). Until now, very little has been published concerning the regulation of this gene or its product. Here, we report the DNA sequence of the PRO3 gene and show that its deduced amino acid sequence has substantial similarity to that of P5C reductases in diverse bacterial species, soybean, and humans. We also measured PRO3 gene expression and P5C reductase activity as a function of growth on various nitrogen and carbon sources, the presence of proline or glutamate in the medium, and its sensitivity to proline starvation and the general control system that regulates the expression of many amino acidbiosynthet...